The goal of this project is to identify new genes for inherited susceptibility to breast cancer. DNA capture and massively parallel sequencing technologies will be exploited to identify these genes in a cohort of 1822 families, each with at least four relatives with breast cancer. The genetic causes of breast cancer in more than 1500 of these families remain unresolved. Discovery of new breast cancer genes in these families involves three aims. In AIM 1, all known genes for inherited predisposition to breast cancer will be fully sequenced in all affected relatives from 150 of the families. The probands of all unresolved families have wildtype sequences at BRCA1, BRCA2, CHEK2, PALB2, CHEK2, BRIP1, p53, and PTEN. However, other affected family members may carry mutations in these genes, and the proband may be a sporadic case. Constitutional DNA of affected relatives will be used to prepare paired-end libraries, which will be hybridized to custom oligonucleotide pools to capture all known breast cancer susceptibility genes, then the enriched libraries will be bar-coded and sequenced in a multiplex design on an Illumina GAIIx genome analyzer. Mutations will be validated by Sanger sequencing. In AIM 2, the Illumina platform will be used again, to fully sequence the entire exomes of all women in the screen with wildtype sequences at all known breast cancer genes. Variants will be filtered to identify rare nonsense mutations, frameshifts, splice site mutations, and genomic deletions and duplications that disrupt genes. Filtered mutations will be validated and tested for co-inheritance with breast cancer. Multiple deleterious mutations are a hallmark of all known genes for inherited breast cancer and are the strongest proof of functional consequence of new breast cancer genes. In Aim 3A, the most promising candidate genes will be fully sequenced in DNA samples from an independent series of familial breast cancer patients to reveal additional mutations. In Aim 3B, potentially functional mutations in these genes will be genotyped by TaqMan assays in large follow-up series to estimate individual and gene-wide mutation frequencies and relative risks. Discovery of new breast cancer susceptibility genes will allow prevention strategies to be extended to families for which causal genes are currently unknown, to better identify women at risk, to allow closer medical surveillance of these women, to stimulate design of new prevention strategies based on genetic evidence, and to offer a better understanding of the biological pathways involved in breast cancer development.